Chimeric oncogenic interferon regulatory factor-2 (IRF-2): Degradation products are biologically active
Rath, Pramod C.
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Interferon Regulatory Factor-2 (IRF-2) belongs to IRF family, was identified as a mammalian transcrip-tion factor involved in Interferon beta (IFNβ) gene regulation. Besides that IRF-2 is involved in immu-nomodulation, hematopoietic differentiation, cell cy- cle regulation and oncogenesis. We have done mo-lecular sub-cloning and expression of recombinant murine IRF-2 as GST (Glutathione-S-Transferase)- IRF-2 fusion protein in E. coli/XL-1blue cells. Re- combinant IRF-2 with GST moiety at N-terminus expressed as GST-IRF-2 (~66 kd) in E. coli along with different low molecular mass degradation products revealed approximately 30, 42, 60 and 62 kd by SDS- PAGE and Western blot, respectively. We further confirm that degradation takes place at C-terminus of the fusion protein not at N-terminus as anti-GST an-tibody was detecting all bands in the immunoblot. The recombinant IRF-2 was bi ologically active along with their degradation products in terms of their DNA binding activity as assessed by Electrophoreti-cally Mobility Shift Assay (EMSA). We observed three different molecular mass DNA/protein com- plexes (1 - 3) with Virus Response Element (VRE) derived from human Interferon IFNβ gene and five different molecular mass complexes (1 - 5) with IRF-E motif (GAAAGT) 4 in EMSA gel. GST only expressed from empty vector did not bind to these DNA elements. To confirm that the binding is specific, all complexes were competed out completely when challenged with 100-X fold molar excess of IRF-E oligo under cold competition. It means degradation products along with full-length protein are able to interact with VREβ as well as IRF-E motif. This means degradation products may regulate the target gene (s) activation/repression via interacting with VRE/ IRF-E.